The detection of specific nucleic acid sequences present in a biological sample is important for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying genetic susceptibility to disease, and measuring response to various types of treatment. Common techniques for detecting and quantitating specific nucleic acid sequences are nucleic acid hybridization and target amplification.
Various hybridization methods are available for the detection and study of nucleic acids. In a traditional hybridization method, the nucleic acids to be identified are either in a solution or affixed to a solid carrier. The nucleic acids are detected using labeled nucleic acid probes which are capable of hybridizing to the nucleic acids. Recently, new hybridization methods have been developed to increase the sensitivity and specificity of detection. One example is the Hybrid Capture® method described in U.S. application Ser. No. 07/792,585 and U.S. Pat. No. 6,228,578. Although these new hybridization methods offer significant improvements over the traditional methods, they still lack the ability to fully discriminate between highly homologous nucleic acid sequences.
The polymerase chain reaction (PCR) is the most commonly used target nucleic acid amplification method. However, PCR is limited to some extent when a large number of different targets are to be amplified simultaneously, i.e., multiplex reactions, which may cause not only PCR artifacts such as primer-dimers, but also spurious target amplification. In an attempt to overcome this limitation, consensus primers may be used when a number of targets have homologous regions. However, homology between species is never 100%, and as a result, the primers will have several mismatches with different targets, causing non-uniform amplification. When different amounts of targets are present in a sample, the amplification efficiency of different PCRs also varies, leading to non-uniform and non-specific amplification of different targets.
It is therefore an object of the present invention to provide a method for detecting target nucleic acid sequences which not only provides increased rapidity and sensitivity, but which is also highly specific and capable of discriminating between multiple highly homologous nucleic acid target sequences